An integrative view on the systematic position of the cupressophyte Cephalotaxus

Abstract We made an in‐depth review of historical studies of the cupressophyte conifer genus Cephalotaxus Siebold & Zucc. with an emphasis on its systematic position. We suggest that the systematic position of the genus is better understood using an integrative approach, so the evolution of phenetic characters is discussed within the context of recent phylogenomics. We propose that the genus should be classified as a separate family Cephalotaxaceae belonging to the clade consisting of Cupressaceae, Cephalotaxaceae, and Taxaceae; the family Cephalotaxaceae is sister to the Taxaceae but not nested within the Taxaceae and is characterized by a unique set of characters including morphology, anatomy, embryology, and chemistry. The family Cephalotaxaceae shows transitional characters between the Cupressaceae and the Taxaceae; the family possesses female cones with a primary cone axis bearing 5–8 pairs of decussate bracts, which is similar to the typical female cones of the Cupressaceae, on the one hand, and may have given rise to the reduced female cone of the Taxaceae with one terminal ovule partially or completely enclosed in a fleshy aril. In parallel, the compound male cone of the Cephalotaxaceae evolved into the seemingly “simple” male cones of the Taxaceae by means of reduction, elimination, and fusion.

microsporangia (Eckenwalder, 2009;Page, 1990). Female organs are pedunculate single or in groups of 2-8 on separate scaly peduncles in axils of bracts ( Figure 1d). The female cone consists of a fleshy cone axis with 3-8 pairs of decussate bracts each having two axillary ovules except for the proximal pair. The ovule secretes a pollination drop containing proteins that are important in defense of nutrientrich pollination drops from microbial pathogens, promotion and support of pollen tube growth, polysaccharide metabolism during drop production and response to stress (Pirone-Davies et al., 2016).
The plum yew genus contains 7-10 extant species that are restricted to subtropical Asia including East Asia and northern Indo-Chinese peninsula (Farjon, 2017;Fu, 1984;Fu et al., 1999;Tripp, 1995;Wang et al., 2022). The genus has a long evolutionary history, fossils ascribed to the genus already occurred in the Early Cretaceous, and fossil species of Cephalotaxus were once widely distributed throughout the Northern Hemisphere in the Cenozoic (Manchester et al., 2009;Shi et al., 2010;Zhang et al., 2019). The modern restricted distribution of the genus is thus clearly relictual.
Systematic relationships of Cephalotaxus Siebold & Zucc. within conifers have been controversial. Some authors included the genus in the Taxaceae (e.g., Christenhusz et al., 2011); some treated it as a separate family, viz. Cephalotaxaceae, either in the suborder Taxineae (Keng, 1975), in the order Taxales (Pulle, 1937), or in the order Coniferae/Coniferales (Pilger, 1926;Takhtajan, 1953), or in the order Cupressales , or as a separate order Cephalotaxales (e.g., Cheng & Fu, 1978). Multiple disciplinary studies were conducted in the last century, but no comprehensive study of the genus has been undertaken. This study is aimed to assess the historical studies of Cephalotaxus and make an integrative study to better understand the systematic position.

| MORPHOLOGY AND DE VELOPMENT
Traditionally, conifers were classified into two major groups: (1) one possessing typical female cones that are normally woody but not fleshy and consisting of a primary cone axis with a variable number of seed scale complexes spirally or decussately arranged, for example, Pinaceae, Araucariaceae, Sciadopityaceae, Cupressaceae; (2) another having atypical female cones that are usually fleshy and reduced with oligomerous seed scale complexes, that is, Podocarpaceae, Cephalotaxaceae, and Taxaceae. In the context of phylogenomic results Ran et al., 2018;Stull et al., 2021), the reduced and atypical female cones of conifers were independently derived as an adaptation to zoochorous dispersal, in Podocarpaceae, Cupressaceae, Cephalotaxaceae, and Taxaceae, respectively (Contreras et al., 2017). The structure of the female cone of Cephalotaxaceae does show similarity to some primitive lineages of Cupressaceae, especially the early development of the female cone of Taiwania cryptomerioides Hayata: two orthotropous ovules are axillary to a fertile bract (Farjon & Ortiz Garcia, 2003). A very high diversity of cupressoid macrofossils was found in the early Mesozoic  (Taylor et al., 2008), so the Cephalotaxaceae may have been rooted in one of them. The Taxaceae are conifers with extremely reduced female reproductive organs that have persisted since the Jurassic (Dong et al., 2020. How the very simple uniovulate organ originated has been a point of discussion in conifer evolution (e.g., Sahni, 1920;Florin, 1948;Keng, 1975;Shi & Wang, 1989) and remains a mystery. The Cephalotaxaceae are definitely pivotal in understanding the origin and evolution of the reduced female organs of Taxaceae.
The female reproductive organs of Cephalotaxus (Figure 2a-h) are markedly different from those of the Taxaceae (Figure 3a-e).
In Cephalotaxus, the female organ is a pedunculate spike and consists of a thickened cone axis bearing 3-8 pairs of decussate bracts, each bract with the exception of the proximal pair subtending two axillary ovules, while a ridge or protuberance is developed between the two ovules ( Figure 2a-c, Lo & Wang, 2001). The axillary two ovules represent a reduced secondary shoot (Wordsell, 1901).
The female cone of Cephalotaxus is thus biaxial and compound (Wordsell, 1901), and close to the typical female cones of the Cupressaceae. In the Taxaceae, the ovule/seed is terminal to a twig and possesses a fleshy aril derived from the fusion of a pair of bracts (e.g., Pseudotaxus W.C. Cheng, Dörken et al., 2019), and thus a uniaxial structure and different from the compound female cones of other conifers. Florin (1951aFlorin ( , 1951bFlorin ( , 1954 proposed the well-known seed scale complex evolutionary model and suggested that conifers are characterized by compound female cones possessing a cone axis with a number of seed scale complexes, the seed scale complex of conifers originated from the spike-like secondary reproductive shoot in Cordaitales by means of evolutionary changes including shoot symmetry from radial to flattened, aggregation of fertile organs to the adaxial side and sterile foliar organs to the abaxial side, and reduction and fusion of the foliar and axial structures. In the female cones of Cephalotaxus, a fertile bract and its axillary two ovules constitute a unit (seed scale complex), which is decussately positioned along the cone axis (Figure 2a,b, Chen, 1999;Lo & Wang, 2001).
Vascular anatomy gives some clues in this respect. In conifers, a foliar organ normally receives one vascular bundle, while an axial organ receives two vascular bundles (Chen, 1999). There are three vascular bundles entering the base of a fertile bract and its axillary two ovules, the fertile bract receiving one bundle and each ovule receiving one bundle, suggesting that the bract is foliar in nature and the two ovules represent two foliar megasporophylls of the secondary axis (Chen, 1999). The ridge/protuberance between the two ovules ( Figure 2b) may be the residual apex of the secondary shoot (Chen, 1999;Lo & Wang, 2001). Stützel and Röwekamp (1999)   the bract of the Ephedraceae, and the outer envelope of Gnetaceae (Bierhorst, 1971;Nigris et al., 2021). It is suggested that fleshy structures of gymnosperms are associated with ovule protection and seed dispersal (Nigris et al., 2021). Some people believed that the reduced fleshy female cones of conifers were derived because of common ancestry (e.g., Keng, 1975). However, molecular phylogenetic studies based on DNA sequences have consistently suggested that the fleshiness of conifers actually evolved multiple times, in Podocarpaceae, Junipers, Cephalotaxaceae, and Taxaceae, respectively (Nigris et al., 2021).
Different opinions exist on the origin of the fleshy part related to seeds of Cephalotaxus. Cheng and Fu (1978) suggested that the succulent part is derived from the basal cushion-like receptacle below the ovule. If this is the case, then the fleshy aril in Cephalotaxus is similar to the cup-like aril in the Taxaceae. Eckenwalder (2009) and Yang et al. (2011) that the seed coat of Cephalotaxus at maturity is differentiated into three layers, viz. endotesta, sclerotesta, and sarcotesta; the sarcotesta is thus different from the aril in the Taxaceae in origin, the fleshy layer of the Cephalotaxaceae being part of the seed coat (developing from integument), while the aril of the Taxaceae is derived from a modification of bracts, for example, Pseudotaxus (Dörken et al., 2019).
Male cones are clustered in globose groups in the axil of leaves in the family Cephalotaxaceae and are thus markedly different from the simple male cones with microsporophylls inserted along the cone axis in the Taxaceae. Florin (1948) suggested that the radial perisporangiate microsporophylls are more primitive than the dorsiventral hyposporangiate microsporophylls in the Taxaceae. Morphological and developmental studies have suggested the derivation of the "simple" male cones of Taxus and Torreya from the ancestral compound cluster of male cones of the Cephalotaxaceae by means of a few evolutionary steps (Dörken et al., 2011;Mundry & Mundry, 2001;Wilde, 1975). At the beginning of this evolutionary process, the lateral cones of a compound male cone cluster in Cephalotaxaceae were reduced to a perisporangiate sporangiophore, for example, in Taxus (Dörken et al., 2011;Mundry & Mundry, 2001;Wilde, 1975).
Then, the adaxial sporangia of the perisporangiate sporangiophore got further reduced to give rise to the hyposporangiate (dorsiventral) sporangiophore in Torreya (Dörken et al., 2011;Mundry & Mundry, 2001;Schulz et al., 2014;Wilde, 1975). As a result, the microsporangium-bearing structure of the Taxaceae is not foliar but axial and equivalent to a pseudanthium (Dörken et al., 2011;Mundry & Mundry, 2001;Schulz et al., 2014). Pseudotaxus chienii (W.C. Cheng) W.C. Cheng occupies a transitional position in this evolutionary process (Dörken et al., 2011). According to this reduction and alteration hypothesis, the family Cephalotaxaceae can be considered as the outgroup of the Taxaceae, and as in the female reproductive organs, the male reproductive organs are also extremely reduced in the family Taxaceae. Pollen morphology also supports the Cephalotaxaceae being more primitive than the Taxaceae (Xi, 1993).

| ANATOMY
The Cephalotaxaceae are similar to the Taxaceae in wood anatomy, for example, the growth rings are inconspicuous, the axial tracheids have spiral thickening, the horizontal wall of wood ray tracheids possess simple pits, the cross-field pits being either piceoid or cupressoid, and also in the type, distribution, and arrangement of stomata, and structure of leaves (Hu, 1999). In addition, both possess transfusion tissue, a kind of tissue occurring only in gymnosperms.
It lies between leaf veins and vascular bundle sheath or endodermis (Hu, 1999). Seven types of transfusion tissue have been recognized in gymnosperms: Pinus type, Pseudotsuga type, Tsuga type, Cupressus type, Taxus type, Araucaria type, and Cycas type (Hu, 1999). The transfusion tissue of the Cephalotaxaceae belongs to the Taxus type and is similar to that in the Taxaceae (Hu & Yao, 1981). However, the Cephalotaxaceae can be distinguished from the Taxaceae by the vascular anatomy of the female cones, the ovule of Cephalotaxaceae being supplied by two inverted bundles, whereas the ovule of the Taxaceae is supplied by a variable number of normally oriented bundles (Singh, 1978). Yadav et al. (2013) suggested that the presence of axial parenchyma is different in Taxaceae and Cephalotaxaceae, being absent in the former but present in the latter family. It is worth mentioning that Cephalotaxus is similar to Torreya and Amentotaxus in the presence of resin canals in leaves on the one hand, and similar to Austrotaxus, Pseudotaxus, and Taxus in lacking vascular sclereids on the other hand (Elpe et al., 2018). These characters make sense in a phylogenetic context that Cephalotaxus is sister to the Taxaceae. Singh (1961Singh ( , 1978 suggested that embryological characters do not support Florin's opinion of the isolated position of Cephalotaxus among the conifers, the unwinged pollen and the absence of prothallial cells in male gametophyte are similar to those of cupressads, taxads, and taxodiads (i.e., Cupressidae). The lack of prothallial cells in the male gametophyte of Cephalotaxus was already noticed by Lawson (1907). Chen and Wang (1990)  However, it is notable that there are a number of embryological distinctions between the two families. Pavement tissue is present in ovules of Cephalotaxaceae but absent in Taxaceae (Singh, 1978).

| EMB RYOLOGY
Archegonia are long, narrow, and pointed at the chalazal end in Cephalotaxaceae but short and blunt in Taxaceae (Singh, 1978).
There are 16 free nuclei in the proembryo of the Cephalotaxaceae (Li et al., 1986) but only eight free nuclei i n that of the Taxaceae (Singh, 1978). The young embryo possesses prominent cap cells in the Cephalotaxaceae, while these are absent in the Taxaceae (Singh, 1978). Chen and Wang (1990) stated that the number of free nuclei of the female gametophyte shows the most remarkable difference between the Cephalotaxaceae and Taxaceae, there being 1024 to 4096 free nuclei before wall formation in the Cephalotaxaceae but only 256 in Taxaceae. They thus concluded that the family Cephalotaxaceae is more primitive than the Taxaceae.
It is general in gymnosperms that the development of the microgametophyte finally gives rise to two sperms. The relative size of the two sperms is variable in the Cupressidae and may have evolutionary significance. The two sperms of Cupressaceae are equal in size, which is a primitive feature (Chen & Wang, 1990). In the Cephalotaxaceae, the two sperms are more or less unequal in size (Chen et al., 1987;Li et al., 1986). In the Taxaceae, the two sperms are very different in size, one is much larger than the other, and only the big sperm is functional (Chen et al., 1987). Chen and Wang (1990) argued that the evolutionary trend in sperm morphology is from equal to unequal in size in conifers, with the Cephalotaxaceae showing a transitional stage in the differentiation of the two sperms between Cupressaceae and Taxaceae. However, it should be noted that Anderson and Owens (1999) reported that the two sperms in Taxus brevifolia Nutt.
are of equal size, a finding corroborated by Wang et al. (2008) in T. yunnanensis W.C. Cheng et L.K. Fu (=T. wallichiana Zucc.). As a result, it remains unclear whether the two sperms are of equal size or not; the evolutionary trend suggested by Chen and Wang (1990) needs to be verified with further embryological observations. It is well-known that Cephalotaxus contains alkaloids and flavones (including biflavones), these chemicals possess taxonomic value. Biflavones have been found in the Cephalotaxaceae and most other conifer families excepting Pinaceae (Hao et al., 2021;Ma et al., 1990;Ma & He, 1999;Mei et al., 2006;Ren et al., 2018), supporting the separation of conifers into two clades, Pinidae (including only Pinaceae) and Cupressidae (including Araucariaceae, Podocarpaceae, Sciadopityaceae, Cupressaceae, Cephalotaxaceae, and Taxaceae).

| CHEMI S TRY
Alkaloids constitute the second major chemical constituent in the Cephalotaxaceae. More than 30 kinds of alkaloids have been isolated and identified so far (Zhu & Zhu, 1999). These alkaloids are mainly classified into two groups, that is, cephalotaxine and homoerythrine-type alkaloids (Chu, 1979;Hao et al., 2021;Mei et al., 2006;Zhu & Zhu, 1999). The Cephalotaxaceae are distinguished from the Taxaceae by the types of the alkaloids. The Taxaceae possess no cephalotaxine and homoerythrine-type alkaloids but have taxine and pseudoephedrine (Chu, 1979;Zhu & Zhu, 1999), which corroborates the taxonomic separation of Cephalotaxaceae from Taxaceae.
The plum yews are economically important not only because of the excellent timber utilized for construction and furniture (Wang & Wang, 1992) but because plants of Cephalotaxus contain important alkaloids, for example, cephalotaxines (Wang & Wang, 1992). The characteristic chemical components of Cephalotaxus including cephalotaxines were found to have important pharmacological effects and could be used to treat patients with acute and chronic diseases such as leukemia and lupus erythematosus (Mei et al., 2006;Zhang et al., 2011). To extract the effective chemicals, Cephalotaxus species in China were over-exploited and many wild populations went extinct (Wang & Wang, 1992). Two species, viz. C. lanceolata and C. oliveri are included in the updated list of National Key Protected Wild Plant Species released in September of 2021 (http://www.fores try. gov.cn/main/5461/20210 908/16251 58505 72900.html). A conflict has arisen between the utilization of natural resources and the conservation of wild populations.

| PHYLOG ENY AND PHYLOG ENOMIC S
Robust phylogenies provide a backbone for reasonable taxonomic treatments. All phylogenetic studies based on DNA sequences consistently indicated that the family Cephalotaxaceae together with the Sciadopityaceae, Cupressaceae, and Taxaceae constitute a monophyletic group, viz. Cupressidae (Chaw et al., 1995;Liu et al., 2022;Rai et al., 2008;Ran et al., 2010Ran et al., , 2018Stefanovic et al., 1998;Stull et al., 2021;Wang & Shu, 2000). concluded that Cephalotaxus is not nested within the Taxaceae but is sister to it. Both plastome phylogenomics (Ji et al., 2021) and Phylogenomics using a large number of single-copy nuclear genes Ran et al., 2018;Stull et al., 2021) have indicated that the family Cephalotaxaceae is sister to but not nested within the Taxaceae.

ACK N OWLED G M ENTS
We thank Bing Liu for his kind help on photographs of Cephalotaxus species. This work was supported by the National Natural Science Foundation of China (grant no. 32270217, 31970205) and the Metasequoia fund of Nanjing Forestry University.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that there is no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data used in the study are included in this paper.